Substrate specificities associated with different members of the diverse families of proteolytic enzymes can be attributed in part to different sets of amino acids, within the binding domain, that are utilized by each enzyme family for substrate recognition and catalysis. A rational approach to engineering proteases has been successful for several proteases. A conserved amino acid residue (glycine 166) known from crystallographic data to reside within the binding cleft of subtilisin was changed to one of several different amino acid residues. The resulting enzyme derivatives showed dramatic changes in specificity towards substrates with increasing hydrophobicity and amino acid size (Estell, et al.). Another bacterially encoded serine endopeptidase, .alpha.-lytic protease, has also been rationally engineered, changing methionine 192 to an alanine. The resulting alteration within the active site of the enzyme appears to have increased structural flexibility of the enzyme active site. The resulting .alpha.-lytic protease derivative has a broader substrate specificity towards larger, more hydrophobic targets (Bone, et al.).
Although these rational approaches have met with success in the altering of substrate specificity, not all mutations effecting substrate specificity are associated with the known or predicted binding cleft of a given enzyme. The substrate specificity of the serine protease trypsin was altered to a chymotrypsin-like function by alteration of amino acids within the binding domain as well as residues known to be outside of the binding domain (Hedstrom, et al.). Mutations outside of the binding cleft of an enzyme can have a profound effect on amino acid residue packing, conformation strain and conformational charge distribution of residues within the binding cleft and as a result can have a profound effect on substrate recognition, catalysis and enzyme stability.
Several other enzymes have also been rationally modified to new substrate-specificities, including T7 DNA polymerase (Ikeda, et al.), lactate dehydrogenase (Wilks, et al.). Finally, natural derivatives of the antibiotic resistance determinant, .alpha.-lactamase, have been obtained as a result of positive selective pressures to novel substrate specificities. There is a precedence for both rational approaches to altering the substrate-specificities of many different enzymes through a detailed understanding of the conformation and biochemical properties of an enzyme. Furthermore, random events which translate into unique enzymatic functions can also be generated under conditions where the proper selective pressures are applied for a desired catalytic function or substrate specificity.